• Introduction:

    • Resources for the study of highly pathogenic micro-organisms are sparsely spread over Europe and even over the world. Also culture collections containing large quantities of these highly pathogenic strains are sparsely distributed, mainly because they are highly demanding to handle. Moreover, most of these pathogens have been identified as agents that have potential to be used as biological weapons. Many emerging questions have been raised after the Anthrax attack in the United States. States (all over the world) were not at all prepared to handle either cases or the large quantities of analyses that were required. Also in Europe, many analyses have been performed, and most of the laboratories involved were not prepared to handle such large quantities or the specific nature of the materials involved. Fortunately nothing infectious was found in Europe. One major conclusion was that first there were no ready diagnostics available for the detection of such pathogens, and since these agents are very infrequent, general knowledge on these organisms is sparse.

    Diagnostic possibilities for detecting these highly pathogenic microorganisms are generally focussed at one, possibly some species. It never passed the border of eg. Prokaryotes. However, in the case of an undefined sample, like a “white powder” sample, the circumscription given to a potential biological weapon, it would be very useful to have a test that is capable of detecting viruses, prokaryotes and eukaryotes (fungi). Therefore a close collaboration between the different microbiologists is necessary. Moreover, such a diagnostic test needs the full characterisation of the microorganisms. Therefor a large number of specialists in different fields in necessary and the information sould consistently be compiled. Such investigations will also bring a lot more information on several characteristics of these microoraganisms that are related with immunology, antibiotic resistance, virulence,…

    Through COST, possibilities will be created for collaborations between laboratories capable of handling dangerous pathogens and laboratories, specialised in the technical aspects of working with the inactivated fractions of the pathogens. It is also necessary to involve both veterinary, public health and defence laboratories, all with their specific tasks within the study of these pathogens.

  • Administration:
    COST is one of the longest-running instruments supporting co-operation among scientists and researchers across Europe. COST now has 35 member countries and enables scientists to collaborate in a wide spectrum of activities in research and technology.

    COST is an intergovernmental network which is scientifically completely self-sufficient with nine scientific COST Domain Committees formed by some of the most outstanding scientists of the European scientific community. The scientific quality control is the main responsibility of the COST scientific Domain Committees which follow very rigorous evaluation procedures established by the COST Committee of Senior Officials (CSO) involving the mandatory use of external peer reviewers.

    To join the COST Action several possibilities are available:

    Depending on your own position and that of the Action there are series of different forms of co-operation.
    1. If your laboratory or institution is in a COST member country (this has nothing to do with your own nationality) you can join the Action as
      1. Member of the Management Committee of an Action in case your country has not yet nominated 2 scientists but is already signatory of the Action. Therefore you have to contact your COST National Coordinator (CNC) who can officially nominate you as representative of your country to the MC committee. It is preferred that you also contact the chair of the Action about your interest.
      2. Member of the Management Committee if your country is not yet signatory of the Action. In that case your country has first to sign its participation in the relevant Action. (Note: If your country wishes to become member 12 months after the approval of the Action the approval of the Management Committee of the Action is required). Here you have to contact your COST National Coordinator (CNC) who can launch the participation of your country in the relevant Action. He will analyze whether the national funding for the Actions activity is there. The CNC can then launch the official participation of your country in the relevant COST Action and he can officially nominate you as representative of your country to the MC committee. In case of an established COST Action (more than 12 months after CSO there is also the agreement of the MC committee required. In any case it is preferred that you contact the chair of the Action about your interest.
      3. Member of a working group of an Action Here you can joint by contacting the chair of the Action (directly or via a colleague from the Action or via the |COST Office) and obtain the invitation to participate in the suitable COST working group.
      4. Invited expert to specific events of an Action (Meetings of the MC, WG, workshop participation etc.) Also her you can join by contacting the chair of the Action (directly or via a colleague from the Action or via the |COST Office) and obtain the invitation to participate in a specific event of the Action.
    2. If your Institution is in a non-COST member state there is the possibility
      1. to participate from an Institution with the status of "non-COST participating Institution" if this was recognized by the Committee of Senior Officals (CSO) as in the mutual benefit.. This allows to participate in the MC and WG meetings although without voting right The participants from non-COST countries are normally not eligible for reimbursement of travel expenses although for specific countries exceptions may be possible.
      2. To participate as invited expert to specific events of an Action (Meetings of the MC, WG, workshop participation etc.)

  • Summary of the project:
    1. Identification of target genes or gene products for the development of specific diagnostic and characterization tools
      • Genomic approach
        In the genomic approach, the project will enlarge identification of unique fragments of DNA of specific microorganisms. In order to identify specific sequences to be used for the development of specific diagnostic tests, several genetic markers will be sought by “differential array”. These fragments, when identified, will also help to define unique fragments for the development of antimicrobial and vaccine targets. Genomes of two different organisms, although closely related, will be hybridised one to another and to themselves. Whole genome libraries will be constructed and the inserted fragments of the library clones will be amplified by PCR and spotted on micro-array slides. Slides will be hybridised with a randomly labelled DNA probe prepared from fragmented genomic DNA, isolated from the organism from which the slides were prepared, and with a probe prepared from a closely related organism. Spotted library fragments that hybridise only with the homologue probe, and not with the probe prepared from the closely related organism. These fragments will be identified in the library and sequenced. The sequences will be compared to known sequences in publicly accessible databases (BLAST search). This will prove the specificity of the fragments. It is expected that several of the fragments obtained will not be present in other species, and likewise will prove their specificity. Random priming can be performed on the organism to have the fragment labelled for detection on the array. In the first instance an inventory of the actual data will be generated, and the consortium will fill gaps within this. A second approach on DNA arrays will be specific for bacteria and fungi. In bacteria, several conserved regions exist to which (degenerate) primers can be constructed. By applying more or less stringent PCR outwards from these genes, fragments of different lengths (intergenic regions) can be produced. Intergenic spacers between the tRNA encoding genes, have been shown to be variable in length. Another possible target is the spacer region between 16S and 23SrRNA genes. For fungi, a similar method can be applied but for these organisms Internally Transcribed Spacer regions ITS1 (between 18S and 5.8S rRNA) and ITS2 (between 5.8 and 25S rRNA) can be used. Based on new and existing data on the length of the fragments, fragments will be chosen and a simple reverse line blot hybridisation will be performed for their possible cross hybridisations. To be able to encompass the discriminatory power of the obtained fragments, several fragments will need to be sequenced to determine their specificity. These sequences will generate further data for post-genomics. This development will need a close collaboration between microbiologists, genomics and groups specialised in technological developments. The major output will directly serve a detection test and will give study material for post-genomics.
      • Post-genomics: antigenic, proteomic and glycomic approach
        Post genomics will enlarge knowledge on the proteins and lipopolysaccharides/glycoproteins in targeted microorganisms as genetic research alone cannot define unique regions encoding proteins, but protein analysis techniques can define such unique proteins. Proteomics aspires to know more than just the identity of proteins, its ultimate pursuit is to understand the functions of proteins and cell’s metabolism. After a protein is produced by the gene-to-amino acid process, it is altered in a number of ways (by posttranslation modifications, glycosylations, phosphorylations, sulphatations, etc.) prior to it being used by the cell. Besides whole cell lysates, purified integral membrane proteins and secreted proteins will be analysed. Using 2D gel electrophoresis combined with mass spectrometry the bacterial antigens that are differentially expressed in strains can be identified. Monoclonal antibodies against selected proteins will be raised in order to define the specificity of these proteins for a microorganism. Characterization and sequencing of the selected peptides, proteins or protein fragments by MALDI-TOF- and LC-ESI-MS/MS will open the field for the preparation of the target peptides, proteins or protein fragments by conventional recombinant techniques. The proteomic results in combination with the immuno-approach will be the basis for the utilization of recombinant techniques for the construction of immune-based prophylactic and/or therapeutic tools exploiting both broad-spectrum and pathogen-specific microbial components. Moreover they can be used in the multiplexed detection assay using flow cytometry. LPSs of pathogenic microorganisms have been considered to be major determinants of virulence expression and infection. They play an important role in interaction of the microbes with hosts, their pathogenicity, immunogenicity, and are capable of inducing antibody responses. Like proteins, the localization and characterization of immunoreactive epitopes will be investigated in the LPSs by a combination of chemical and immunological methods. The next step includes characterization and sequencing of the selected, poly- and oligomeric glycoses and their fragments, obtained from the native LPSs, by MALDI-TOF- and EIS-MS techniques.
    2. Technology development
      The technological developments in this project enable further characterisation of the microorganisms covered by this proposal. Two technological platforms will be used for the diagnostic assay development: DNA-microarray Specific capture probes designed on the basis of the genomic research described above will be spotted on chips. After an amplification step (or preferably not) the sample material hybridised to this probe will be revealed by a labelled DNA probe. Different kinds of sample preparation, and signal amplification methods will be tested in order to achieve maximum analytical sensitivity. The final goal is to have as few as possible preparation protocols for the different types of sample that could be analysed. Suspension microarray (DNA/Antibody/protein/LPS, etc.) Suspension arrays of microspheres, analysed using flow cytometry, offer a new approach to multiplexed assays for large-scale screening applications. By optically encoding micron-sized polymer particles, suspension microarrays can be created to enable highly multiplexed analysis of complex samples. These multiple fluorescent microspheres, conjugated to different probes (DNA, Lipid, proteins, LPS or their fragments), constitute the solid phase for detecting the presence of a biological agent’s gene, antigen or protein in samples.
    3. Microbiology
      The bacterial pathogens that will be studied are Bacillus anthracis, Brucella spp., Burkholderia spp. (B.mallei & B.pseudomallei), Coxiella burnetti (and closely related Rickettsiae), Yersinia pestis (plus Y. pseudotuberculosis), Francisella tularensis, and Clostridium botulinum. The yeasts and filamentous fungi under investigation will be Aspergillus fumigatus and A. flavus, Blastomyces dermatitidis, Candida albicans, Cladophialophora bantiana and C. devriesii, Coccidioides immitis and C. posadasii, Cryptococcus gatti and C. neoformans, Exophiala dermatitidis, Fusarium verticillioides, Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, Pseudallescheria boydii, and Ramichloridium mackenziei. Viruses from the RNA virus families Bunyaviridae, Filoviridae, Arenaviridae, Togaviridae, Paramyxoviridae, Orthomyxoviridae and from the DNA virus family of the Poxviridae are prime candidates. All these microorganisms need special culture and growth conditions. They all have to be treated in high containment environments like BSL3 and BSL4 conditions. Highly specialised skills are necessary to safely treat these microorganisms. Several laboratories that have high containment facilities and who are used to working with these highly pathogenic microorganisms subscribe to this COST Action. In order to evaluate the usefulness of the developed techniques, validation is necessary. Evaluation of the analytical test characteristics of each individual system will be done with the help of antigen or DNA extracts prepared from pure cultures or protein production at known quantity/concentration and with a panel of relevant biological agents that are known to interfere in other types of diagnostic assays. Once these steps of the analytical evaluation have been shown to be successful, the test characteristics of the different multiplexed detection systems will be evaluated and compared with more conventional approaches, brought by microbiologists, who are used working with the microorganisms in this study.

  • Chair,vice chair,organisation:
    • Chair:
      Prof. Dr. P. Butaye, DVM, PhD
      Department of Bacteriology and Immunology
      Veterinary and Agrochemical Research Centre
      VAR-CODA-CERVA
      Groeselenberg 99
      B-1180 Ukkel
      Belgium

      Department of Pathology, Bacteriology and Poultry Diseases
      Faculty of Veterinary Medicine
      Ghent University
      Salisburylaan 133
      9820 Merelbeke
      Belgium

      Tel: + 32 2 379 0415
      Fax: + 32 2 379 0670
      Mobile: + 32 474 982 339
      E-mail: pabut@var.fgov.be
      alternate E-mail: pabuta@google.com

    • Vice Chair:
      Dr. Rudolf TOMAN
      Laboratory for Diagnosis and Prevention
      of Rickettsial and Chlamidial Infections
      Institute of Virology
      Slovak Academy of Sciences
      Dubravska cesta 9
      SK – 845 05 Bratislava
      Slovakia
      Tel: +421 2 593 02418
      Fax: +421 2 547 4284
      E-mail: Virutoma@savba.sk

      The chair may always be contacted for further information on this COST Action B28.

    • Organisation:
      The MC (Management committee) will coordinate all research initiatives to be complementary and additional for the final research aims. Within the 5 WGs (Working group), described underneath, the work should be equally and fairly divided among its members. WP chairs will be in charge of this, WG chairs will be part of MC. MC meetings will be organised at least twice a year together with the WG meetings. In collaboration with the WG leader, MC can decide to have an additional separate WG meeting.
      A website with a restricted access area will be created in order to improve data exchange between the different partners of the COST Action.
      Great attention will be put STSM, since techniques applied in this Action are highly specialised. Appraisal of such techniques request specialised training. A committee deciding on the candidates that will be selected will be chosen from the MC. After they have finished their mission, their results will be orally presented to a MC/WG meeting. A diagram is representing the interdependencies of the different WGs.